Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Published online 26 March 2008 Nucleic Acids Research, 2008, Vol. 36, No. 8 2739–2755 doi:10.1093/nar/gkn030 Structure of the DNA-binding domain of ...
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Published online 26 March 2008

Nucleic Acids Research, 2008, Vol. 36, No. 8 2739–2755 doi:10.1093/nar/gkn030

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins Sunggeon Ko1,3,y, Sung-Hoon Jun2,y, Hansol Bae2, Jung-Sue Byun2,3, Woong Han1,3, Heeyoung Park1,3, Seong Wook Yang2, Sam-Yong Park4, Young Ho Jeon5, Chaejoon Cheong5, Woo Taek Kim2,*, Weontae Lee1,3,* and Hyun-Soo Cho2,3,* 1

Received September 2, 2007; Revised January 17, 2008; Accepted January 18, 2008

ABSTRACT Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNAbinding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1561–681), and was composed of 4 a-helices. We also determined the structure of NgTRF1561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1561–681 with the DNA-binding domain of hTRF1. The interaction

of Arg-638 with the telomeric DNA, which is unique in NgTRF1561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)n. INTRODUCTION Telomeres are essential for eukaryotic genome stability (1). During the last decade, telomeres have been the subject of intense study because of the link between telomere function and cancer and aging (2,3). Telomeric DNA consists of tandem repeats of simple conserved sequences, and functions in maintaining the integrity of flanking chromosomal sequences during replication (1,4). Telomeric DNA that is shortened during replication is restored through the action of telomerase, a reverse-transcriptase that synthesizes telomeric DNA using its own RNA molecule as a template (5,6). The synthesis of telomeres by telomerase and telomere length is regulated by numerous telomere-binding proteins. While the function of telomere-binding proteins in the regulation of telomere length is well characterized (7), other functions of them have also been described. The telomere-binding protein complex enables the DNA repair machinery to distinguish telomere ends from doublestranded DNA breaks. Defects in the telomere-binding protein complex trigger DNA damage response pathways that arrest the cell cycle and activate cell senescence or

*To whom correspondence should be addressed. Tel: +82 2 2123 5651; Fax: +82 2 312 5657; Email: [email protected], [email protected], [email protected] y

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Department of Biochemistry, 2Department of Biology, 3Protein Network Research Center, College of Life Sciences and Biotechnology, Yonsei University, Seoul 120-749, Korea, 4Protein Design Laboratory, Yokohama City University, Suehiro 1-7-29, Tsurumi-ku, Yokohama 230-0045, Japan and 5Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883, Korea

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cell division and the cell cycle (27). Overexpression of NgTRF1 resulted in a shorter telomere length compared to wild-type plants, whereas decreased expression of NgTRF1 resulted in a longer telomere length (33). Moreover, these perturbations of the expression of NgTRF1 caused apoptotic cell death. Recently, the in vivo function of rice telomere-binding protein, RTBP1, has been studied at the plant level (34). Loss-offunction (amorphic or hypomorphic) mutants of RTBP1 exhibited defects in both vegetative and reproductive development, and these phenotypes correlated with the gradual acquisition of dysfunctional telomeres. The structures of double-stranded telomere-binding proteins also have been studied mainly in human and yeast (35–38). The structures of full-length telomerebinding proteins have not been reported due to the presence of flexible linker regions within these proteins. Therefore, attention has focused on the structures of the Myb-like DNA-binding domains to understand telomerebinding mechanism. The DNA-binding domains of human double-stranded telomere-binding proteins, hTRF1 and hTRF2, are composed of three a-helices, and form a helix–turn–helix DNA-binding motif (35,37). The complex structure of the Myb-like domain of hTRF1 and telomeric DNA shows that the helix–turn–helix motif recognizes telomeric DNA sequences in the major groove and that the N-terminal region interacts with DNA in the minor groove (35,38). The crystal structure of hTRF2 DNA-binding domain in complex with telomeric DNA shows that it recognizes the same sequence as hTRF1 (35). RAP1 of budding yeast, scRAP1, contains two subdomains that are closely related in structure to Myb domains. The two subdomains are connected by a long linker, and they recognize and bind to two independent tandem telomeric repeats (36). The structure of the DNA-binding domain of Arabidopsis TRP1 (AtTRP1) was recently reported using NMR spectroscopy. It is composed of 4 a-helices suggesting that plant telomerebinding proteins have a unique DNA-binding domain compared to other organisms (39). Chemical shift perturbation assay suggested that helix 3 and the flexible loop connecting helix 3 and helix 4 are involved in the recognition of telomeric DNA sequence. Moreover, telomere DNA sequences have been identified as (TTAGGG)n in majority of eukaryotic organisms but (TTTAGGG)n in plants (40–42). However, the exact overall picture of how plant double-stranded telomerebinding proteins recognize plant telomeric sequence, (TTTAGGG)n, and the role of plant-specific C-terminal Myb-extension in the telomeric sequence recognition is unclear. To address these questions, we solved the structure of the DNA-binding domain of NgTRF1 in complex with telomeric DNA. The molecular details of the interaction between the DNA-binding domain of NgTRF1 and plant telomeric DNA suggests that the interaction mode of plant and human double-stranded telomere-binding proteins are highly conserved during evolution. However, the plant-specific C-terminal Myb extension is required for the specific recognition by NgTRF1 of the sequence (TTTAGGG)n, which is specific for plant telomeres.

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apoptosis (8–10). Telomere-binding proteins also protect telomeres from inappropriate DNA repair reactions, such as end-to-end joining and exonucleolytic digestion (11). In humans, six telomere-specific proteins have been known to form a complex (12). Of them, three proteins, hTRF1, hTRF2 and hPOT1, directly bind to telomeric DNA sequences and they are interconnected by two additional proteins, hTIN2 and hTPP1. hTRF1 and hTRF2 are double-stranded DNA-binding proteins, while hPOT1 binds to single-stranded DNA. hTRF1 forms homodimers, and possesses a Myb-like domain through which it binds to specific DNA sequences (13–15). The role of hTRF1 in the regulation of telomere length has been demonstrated by gain-of-function studies (7,15) in which overexpression of wild-type allele caused telomere length to shorten and expression of a dominant negative allele resulted in progressive elongation of telomeres, until a new equilibrium was achieved. hTRF2 is a paralog of hTRF1 and its primary function is in telomere capping, which prevents end-to-end joining (11,16,17). hPOT1 has been proposed to function downstream of hTRF1 to relay the negative regulation to the telomere terminus (18). Several telomeric proteins have been identified in yeast. In the budding yeast Saccharomyces cerevisiae, RAP1 (scRAP1) is a doublestranded DNA-binding protein, and the primary telomere-binding protein (19–21). Excess scRAP1 bound at the telomere negatively regulates telomere elongation in cis through the inhibition of telomerase activity (22,23). However, while scRAP1 is functionally analogous to hTRF1, the two proteins are not homologous. In contrast, fission yeast contains an ortholog of hTRF1, TAZ1, which binds to telomeric DNA duplexes and negatively regulates telomere length (24). The biological functions of telomeres and telomerebinding proteins have been studied extensively in humans and yeast. Double- or single-stranded telomere-binding proteins in plants have also been identified, which indicates that this class of proteins has been conserved throughout evolution (25–28). In Arabidopsis, there are at least 12 TRF-like (TRFL) genes that have a single Myb-like domain in their C-terminal region and they fall into two distinct gene families based on the presence or absence of the C-terminal Myb-extension (29). Recombinant TRFL family 1 proteins, which contain C-terminal Myb-extension form homo- and hetero-dimers and specifically interact with plant double-stranded telomeric DNA in vitro. TRFL family 2 proteins lack the C-terminal Myb-extension, similarly to nonplant telomere-binding proteins such as hTRF1 and hTRF2, but they cannot bind to telomeric DNA. Single myb histone (SMH) family proteins, which have a single Myb-like domain in their N-terminal region, also bind telomere DNA repeats in vitro and they are plant specific (30,31). The protein AtTRB1, a member of SMH family, interacts with the Arabidopsis homolog protein of hPOT1, AtPot1, suggesting its plant telomere-specific role (32). The physiological functions of telomere-binding proteins in plant have been studied recently. The expression of NgTRF1, a tobacco double-stranded telomere-binding protein, is regulated tightly in correlation with

Nucleic Acids Research, 2008, Vol. 36, No. 8 2741

NMR spectroscopy

Cloning, protein expression and purification and DNA preparation

The telomere DNA-binding domain of NgTRF1 was prepared in a solution of 90% H2O, 10% D2O or 99.9% D2O, pH 7.0 in 25 mM sodium phosphate buffer, 100 mM NaCl. The purified DNA-binding domain of NgTRF1 was concentrated to 1 mM by centrifugation using an Amicon filter unit (Milipore). All NMR spectra were recorded at 298 K using Bruker DRX500 MHz and DRX600 MHz spectrometers equipped with a tripleresonance inversed probe with x, y, z gradient shielding. A cryoprobe was also used. 1H chemical shifts were referenced directly to internal 4,4-dimethyl-4-silapentane1-sulfonic acid (DSS). 15N and 13C shifts were referenced indirectly. The strong solvent resonance was suppressed by water-gated pulse sequence combined with pulsed-fieldgradient (PFG) pulses. Backbone and Cb resonances were assigned using the following techniques in succession: twodimensional (2D) 1H-15N HSQC, constant-time-1H-13C HSQC and 3D HNCO, HNCACB, CBCA(CO)NH and HNCA spectra. In some experiments, HN(CA)CO and HN(CO)CA were also collected. Side-chain and Ha assignments were obtained using HBHANH, H(CC)(CO)NH-TOCSY, 15N-edited NOESY, 13C-edited NOESY, HCCH-TOCSY and HNHA spectra. As a final step, HCCH-TOCSY was collected after solvent exchange to D2O. Distance restraints for the DNA-binding domain of NgTRF1 were obtained using 15N-edited NOESY and 13C-edited NOESY spectra, with mixing times of 100– 150 ms in 90% H2O, 10% D2O or 99% D2O to extract NOE information. Slowly exchanging amide protons were identified by lyophilizing a fully protonated sample in H2O to dryness, re-dissolving it in a solution of 99.99% D2O, and then acquiring the 2D 1H-15N HSQC spectrum immediately, or after 1 day. 15 N-1H NOE values were calculated as the ratio of the intensities of paired 15N-1H correlation peaks from interleaved spectra acquired with and without 1H presaturation during a recycle time of 5 s. All NMR data were processed using Bruker XWINNMR (Bruker Instruments) and NMRPipe/NMRDraw software (43) on a Linux-operating PC workstation and analyzed (resonance assignments and cross-peak picking/ integration, etc.) using Sparky 3.60 software. In the acquisition dimension the small residual water resonance was removed by a solvent-suppression time domain filter, zero-filled to twice the size and Fourier-transformed. All indirect dimensions were processed using a linear prediction (LP) to enhance resolution. The size of the 15N time domain was doubled by mirror image LP. Forward– backward LP was applied to the 13C and 1H domains. HNCO was used to resolve overlap in 1H-15N HSQC spectra.

DNA fragments encoding the deletion mutants of NgTRF1 were cloned into pPROEX (Invitrogen) for hexa-histidine tagging proteins expression and the plasmid pGEX4T-1 (Amersham Biosciences) for glutathioneS-transferase (GST) fusion proteins expression. For structural studies, hexa-histidine tagging proteins (pPROEX vector) were used. The DNA-binding domain of NgTRF1, NgTRF1561–681, was overexpressed in Escherichia coli BL21 (DE3)-CodonPlus strain (Stratagene). The cells were grown at 378C to an optical density at 600 nm (OD600) of 0.5, then 1 mM isopropyl-1thio-b-D-galactopyranoside (IPTG) was added to induce protein overexpression at 258C. After an additional 8 h of growth, the cells were harvested and subjected to centrifugation. The cell pellets were suspended in 25 mM sodium phosphate (pH 7.0), 100 mM NaCl and sonicated. Recombinant proteins were initially purified by Ni–NTA column (Amersham Biosciences), followed by TEV protease digestion and a second round of Ni–NTA chromatography to remove the fusion tags. Further purification of the protein was carried out using a Superdex 75 gelfiltration column (Amersham Biosciences). For DNA interaction studies using EMSA assay, GST-fusion proteins (pGEX4T-1 vector) were used. Several NgTRF1 mutants (Figure 1) were overexpressed in E. coli BL21 (DE3)-CodonPlus strain (Stratagene). For overexpression of NgTRF1 1 mM IPTG was used when the cell growth reached optical density at 600 nm (OD600) of 0.6 in 378C in shaking incubator. After IPTG induction, the cells were incubated at 258C with gently shaking. After 8–10-h incubation at 258C, the cells were harvested and subjected to centrifugation. When the cells were sonicated in 25 mM sodium phosphate (pH 7.0), 100 mM NaCl and centrifuged to remove cell debris, supernatants was loaded on glutathione-SepharoseTM high performance resin (Amersham Biosciences) and GST-fusion NgTRF1 was purified. For removing GST-tag, thrombin protease (Amersham Biosciences) was used. Superdex 75 gelfiltration column (Amersham Biosciences) was applied to further purification. Complementary strands of a 14mer consisting of two repeats of the telomeric DNA sequence (50 -TTTAGGG TTTAGGG-30 ) were chemically synthesized and solved in distilled water. For annealing, each oligomers of same molar ratio were mixed and put in the 948C Dry-Bath (Barnstead Co., Ltd) for 5 min. The Dry-Bath containing DNA mixture was cooled down slowly in room temperature. To remove additive chemicals from DNA synthesis and do NMR experiments, DNA solution was dialyzed to protein buffer (pH 7.0 in 25 mM sodium phosphate buffer with 100 mM NaCl) using dialysis membrane, MWCO 5 KDa (Spectra/PorÕ dialysis Co., Ltd) for 12 h. 1D NMR experiment in Bruker DRX 500 MHz confirmed doublestrand DNA. DNA concentrations were determined by measuring absorbance at 260 nm.

NMR structural calculations Distance restraints were derived from cross-peaks in N-edited NOESY (m = 100 and 150 ms) and 13 C-edited NOESY (m = 150 ms) spectra. Slowly exchanging amide protons were identified by acquiring a series of 1H-15N HSQC spectra after dissolving lyophilized protein into 100% D2O. Angle constraints were obtained 15

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MATERIALS AND METHODS

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Binding activity to telomere repeat

(a)

Myb

1

NgTRF1 52kDa

GST

26kDa

2 mg 0

52 26 13 12

681

441

681 681

TTTAGGG AAATCCC 2 + + +

573

12kDa (NgTRF1573−681) (b)

441

561

13kDa (NgTRF1561−681)

681

681



4 mg 52 26 13 12 kDa

TTTAGGG AAATCCC 2

Figure 1. Mapping of the DNA-binding domain of NgTRF1. (a) Schematic representation of full-length and deletion mutants of NgTRF1. GST is represented by the shaded box; black boxes represent the Myb-like domain of NgTRF1; regions outside the Myb-like domain are represented by open boxes. The molecular mass of each mutant polypeptide is indicated in the left column. The binding activity of each mutant protein is presented in the right column. (b) Gel retardation assays. Total of 2 or 4 mg of the indicated purified protein were incubated with 0.25 ng of radiolabeled double-stranded telomeric DNA (TTTAGGG)2, and then subjected to electrophoresis on a nondenaturing polyacrylamide gel. Protein–DNA complexes were visualized by autoradiography.

from the TALOS prediction (44). Protein structure was calculating using the CYANA program version 2.1, which combines automated assignment of NOE crosspeaks and structural calculations. Chemical shift tolerances were set at 0.02 p.p.m. for protons, and 0.3 p.p.m. for nitrogen and carbon. Additional tolerances were set at 0.03 p.p.m. and 0.04 p.p.m. for protons and heavy atoms, respectively. NMR-derived experimental restraints contained 1168 unambiguous NOEs [175 intraresidue, 296 sequential, 206 medium-range (2

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